title: Global analysis of FRET-FLIM data in live plant cells.
abstract: This chapter describes the procedure for globally analyzing fluorescence lifetime imaging (FLIM) data for the observation and quantification of Förster resonance energy transfer (FRET) in live plant cells. The procedure is illustrated by means of a case study, for which plant protoplasts were transfected with different visible fluorescent proteins and subsequently imaged using two-photon excitation FLIM. Spatially resolved fluorescence lifetime images were obtained by application of global analysis using the program Glotaran, which is open-source and freely available software. Using this procedure it is possible to extract the fraction and distance of interacting species between, or conformational changes within proteins, from complex experimental FRET-FLIM datasets, even at low signal-to-noise ratios. In addition, the software allows excluding inherently present autofluorescence from the plant cells, which improves the accuracy of the FRET analysis. The results from the case study are presented and interpreted in the context of the current scientific understanding of these biological systems.
authors.affiliation: Laboratoire d'Optique et Biosciences, INSERM U696-CNRS UMR7645, Ecole Polytechnique, Palaiseau, France.
authors.forename: Sergey P
authors.initials: SP
authors.lastname: Laptenok
authors.forename: Joris J
authors.initials: JJ
authors.lastname: Snellenburg
authors.forename: Christoph A
authors.initials: CA
authors.lastname: Bücherl
authors.forename: Kai R
authors.initials: KR
authors.lastname: Konrad
authors.forename: Jan Willem
authors.initials: JW
authors.lastname: Borst
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D061848:Optical Imaging; D059828:Plant Cells / Q000648:ultrastructure*; D011523:Protoplasts / Q000648:ultrastructure; D059629:Signal-To-Noise Ratio; D012984:Software
chemical_list: D049452:Green Fluorescent Proteins